Project Summary Cardiac sarcomeres represent the basic units of contraction driving the beating of the heart. At their core, cardiac sarcomeres are composed of stacks of beta cardiac myosin II (? CMII) which hydrolyze ATP to generate force by pulling on actin filaments. Mutations in ? CMII account for ~40% of all cases of inherited hypertrophic cardiomyopathy (HCM). HCM can lead to arrhythmias, debilitating lifestyle, heart failure, and is the leading cause of death among young adults and athletes. A postmortem hallmark of HCM is sarcomere disarray. Despite their importance, how cardiac sarcomeres are formed and maintained in healthy individuals, and how this is perturbed in disease states is not understood. The lack of a mechanistic understanding of sarcomere formation precludes effective treatment and therapeutics for diseases which affect sarcomere organization, such as HCM. Highlighting this, there are currently no FDA approved drugs that specifically treat HCM. Our lab has recently leveraged super-resolution microscopy to show how non-muscle myosin II (NMII) based contractile systems assemble into large ensembles which resemble cardiac sarcomeres in structure and function (i.e., to generate force). Referred to as NMII stacks, these large ensembles formed via two non- mutually exclusive mechanisms. 1.) NMII stacks grew from an expansion of single NMII filaments via a series of distinct structural steps, and 2.) via concatenation of multiple filaments (i.e., multiple NMII filaments ?running into? each other). Due to their structural and functional similarities, I hypothesize that the mechanisms underlying NMII stack formation are conserved in ? CMII filaments during sarcomere formation. Expanding upon our previous work in ?non-muscle? contractile systems, this project will leverage recent advances in human stem cell technology and super-resolution microscopy techniques to elucidate the mechanisms of cardiac sarcomere formation. Specifically, we will test how ? CMII filaments assemble into larger ? CMII stacks found at the core of sarcomere structures. Our lab has recently developed a live-cell imaging approach which allows us to observe sarcomere formation in live cells. Fluorescently tagged ? CMII will be used in conjunction with this assay to determine if ? CMII stacks form via similar mechanisms as NMIIA stacks. We will also utilize genetic mutants to test if mutations in ? CMII which lead to HCM affect sarcomere formation and/or maintenance. Having established a model of sarcomere formation, we will test the requirement of NMII isoforms during sarcomere formation, as mice lacking NMIIB fail to develop organized sarcomeres, and NMII isoforms are found in ?nascent? sarcomere structures. Together, these experiments will elucidate the mechanisms of cardiac sarcomere formation, and how this is perturbed in disease states which lead to HCM.